293T cells should be split 3 times a week: Use cells that are below passage 15 for viral production. Don't have either application? If pooling harvests, transfer the harvested media to a polypropylene storage tube and store at 4 ℃ between harvest. Dissolve 100 mg of powder in 100 mL of deionized water. & Engineering, Model Aliquots can be thawed and stored at 4 ℃ prior to use. What do I need to know about the customs and importation process for my country? Prepare a mixture of the 3 transfection plasmids: Dilute the above 500 μL mixture into 500 μL PEI-OptiPro SFM with enough PEI such that the ratio of μg DNA:μg PEI is 1:3 (1000 μL total per 10 cm dish). Once produced, lentivirus can be used for a variety of downstream applications such as stable-cell line generation. After 2 months, discard the tube and thaw a new working stock. COVID-19 and Coronavirus Plasmids & Resources page, Genome Fields, Pathways Refer to the table below for a possible range of ratios to test: Figure 1: Lenti-X 293T cells were transfected with the GFP-expression plasmid pRosetta using μg total DNA to μg PEI ratios of 1:1, 1:2, 1:3 and 1:6. & ORFs. The following morning, carefully aspirate the media. For 10 mL of DMEM complete, add 10 μL of 25 mM chloroquine diphosphate. How can I track requests for my plasmids? Learn more, Please note: Your browser does not fully support some of the features used on Addgene's website. What strain of bacteria does my stab contain? lentiCRISPRv2 (one vector system): This plasmid contains two expression cassettes, hSpCas9 and the chimeric guide RNA. Have questions about your order, deposit, or a plasmid? Filter sterilize through a 0.22 μm filter. Left panels: bright field images; right panels: GFP channel images. After thawing, the solution can be stored at 4 ℃ for up to 2 months. Dissolve 0.129 g of chloroquine diphosphate salt in 10 mL of sterile water. Thawed aliquots should be discarded after 1-2 months. While stirring, slowly add hydrochloric acid until the solution clears. Receive the latest news, hot plasmids, discounts and more. The viral supernatant can be stored at 4 ℃ for several hours but should be aliquotted and snap frozen in liquid nitrogen and stored at -80 ℃ as soon as possible to avoid loss of titer. Remove media, replace with fresh media. Map and Sequence File:    Download    Open, Sequence Author:  Zhang Lab / Addgene #52961. Download SnapGene or SnapGene Viewer. © 2020 GSL Biotech LLC | Sitemap | Privacy Policy | Legal Disclaimers. To a 500 mL bottle of DMEM high glucose, add 55 mL of heat inactivated FBS and 11 mL of 200 mM L-alanyl-L-glutamine. LentiCRISPR (pXPR_001): This plasmid contains two expression cassettes, hSpCas9 and the chimeric guide RNA. The vector can be digested using BsmBI, and a pair of annealed oligos can be cloned into the single guide RNA scaffold. Editing, Cloning Once produced, lentivirus can be used for a variety of downstream applications such as stable-cell line generation. Do I need a new MTA for Penn viral vectors? This procedure can be modified for alternative packaging cell lines or transfection reagents. Home » Resources » Plasmid Files » CRISPR Plasmids » lentiCRISPR v2. Typically the solution will be basic and will need adjustment with hydrochloric acid first. The health of the packaging cell line is critical for obtaining high viral titer. Gently aspirate media, add 10 mL fresh DMEM complete containing 25 μM cloroquine diphosphate and incubate ~5 hours. Filter supernatant through a 0.45 μm PES filter. Day 2 (am): 18 hours post transfection. Zhang lab lentiviral transfer plasmid, also known as pLentiCRISPR v2, for CRISPR/Cas9 genome editing of mammalian cells with a single guide RNA (sgRNA). Incubate the cells for 18 hours, or until the following morning. Replace the media with 15 mL of DMEM complete. If you run into any problems registering, depositing, or ordering please contact us at [email protected] By continuing to use this site, you agree to the use of cookies. Systems, Research Please note: Your browser does not support the features used on Addgene's website. Does Addgene accept orders by fax, phone or email? The map, notes, and annotations on this page and in the sequence/map file are copyrighted material. This plasmid is available through Addgene. To see this sequence with restriction sites, features, and translations, please download SnapGene or the free SnapGene Viewer. Allow the solution to mix for 10 min and then recheck the pH to ensure that it has not drifted. ×Please choose an application for opening sequence files. To learn more about how we are supporting COVID-19 research and to find related plasmids, check out our COVID-19 and Coronavirus Plasmids & Resources page. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. To see this sequence with restriction sites, features, and translations, please download SnapGeneor the free SnapGene Viewer. The optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch of 1 mg/mL PEI and for each cell line. Addgene is open for ordering and depositing; find up-to-date details here. Gently add the diluted PEI to the diluted DNA. Filter the solution through a 0.22 μm membrane. Incubate the mixture 15-20 min at room temperature. This protocol can be used to produce lentivirus from a lentiviral vector transfected into Lenti-X 293T cells using a polyethyenimine (PEI) transfection protocol. The 1:2 and 1:3 total DNA:PEI μg ratios provided high transfection efficiencies as measured by the highest proportion of GFP positive cells without limiting cell growth. There is a problem with the plasmid I received. What is an MTA/Who is authorized to sign? This website uses cookies to ensure you get the best experience. This procedure can be modified for alternative packaging cell lines or transfection reagents. This protocol is for creating individual lentiCRISPR targeting a single genomic locus. Centrifuge the viral supernatant at ~500 g for 5 minutes to pellet any packaging cells that were collected during harvesting. Learn about the latest plasmid technologies and research tools. Add the transfection mix dropwise being careful not to dislodge the cells. This protocol can be used to produce lentivirus from a lentiviral vector transfected into Lenti-X 293T cells using a polyethyenimine (PEI) transfection protocol. How can I be notified when a plasmid from a specific lab or paper is available? You may not be able to create an account or request plasmids through this website until you upgrade your browser. Carefully transfer the transfection mix to the Lenti-X 293T packaging cells. Virus can be harvested at 48, 72, and 96 hours post transfection in individual harvests or a combined harvest where all the individual harvests are pooled. Store at 4 ℃. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047. GeneArt CRISPR Nuclease Vector - CD4 (linearized), GeneArt CRISPR Nuclease Vector - OFP (linearized), pHRdSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE, pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s), pU6-(BbsI)_CBh-Cas9-T2A-mcherry-P2A-Ad4E4orf6. Plasmid lentiCRISPR v2 from Dr. Feng Zhang's lab contains the inserts Cas9 and Puromycin resistance and is published in Nat Methods. L-alanyl-L-glutamine (or alternative stable glutamine), Low serum medium such as Opti-MEM or Opti-Pro SFM, DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine. The vector can be digested using BsmBI, and a pair of annealed oligos can … lentiCRISPR v2.dna Map and Sequence File: Download Open This protocol is for creating individual lentiviral CRISPR plasmids targeting a single genomic locus. How do I place an order? Use hydrochloric acid or sodium hydroxide to adjust the pH to 7.0. Learn more, Download our file to copy and paste plasmid data, Open collection of AAV data generously shared by scientists, Basic analysis for a user-entered sequence; includes restriction sites and map, Digital collection of empty plasmid backbones from publications and commercially available sources. What is virus associated DNA, and why do I have to order it?